This invention relates generally to analytical instrumentation for determining macromolecular interaction and is particularly directed to a microcomputer controlled chromatography system employing size exclusion and gel filtration for the qualitative and quantitative analysis of the interaction of macromolecules such as proteins.
The interaction of macromolecules results in the formation of a noncovalently assembled complex that is characterized by a larger Stokes radius than that of either of its constituents. Therefore, ultracentrifugation, inelastic light scattering, and size-exclusion chromatography (SEC), each of which depends upon the Stokes radius of a macromolecule, have been applied in protein-protein association studies. The general availability, conceptual directness, and minimal expense of SEC suggests that this procedure should be a practical approach to many problems of macromolecular interaction analysis if quantitative reproducibility can be ensured. Size-exclusion gel filtration has thus been used to qualitatively study the interaction between two proteins on the basis of a dependence of observed elution positions on sample concentration, wherein the filtration time is dependent on the size of the particles. The formation of a stable, higher molecular weight species by a mixture of two proteins has been observed in the case of high affinity interaction. The Hummel-Dreyer technique has frequently been used for quantitative chromatographic evaluation of interaction by analysis of a small zone of ligand on a column saturated with the binding molecule. Thus, a column saturated with a DNA plasmid has been used to measure the affinity of a restriction enzyme for its nucleic acid binding site. Large zone SEC analyses based on interpretation of the shape of the boundary profile or the median position of the sample plateau of the mixture have been extensively studied.
To conserve potentially rare or irreplaceable biological molecules, however, a small zone gel-chromatography strategy is desirable. Quantitative interpretation of small-zone elution profiles has been hindered by the absence of a definitive mathematical description of the dynamic behavior of a sample of interacting solutes during development of the chromatogram. Approximate solutions have been developed which appear to be appropriate for experimental strategies in which one solute is in sufficient excess to generate pseudo first-order reaction kinetics and have allowed for analysis of interactions observed by size-exclusion high performance liquid chromatography (HPLC). In another approach, an iterative computer simulation of the elution process has shown promise for both qualitative and quantitative interpretation of small zone chromatograms. See F. J. Stevens and M. Schiffer, Biochem. J. 195, 213-219 (1981).
Although HPLC provides an appropriate analytical system for studying the degree of interaction between macromolecules in terms of precision, reproducibility and speed, contemporary silica-based HPLC size-exclusion matrices retain interactions with proteins that preclude the use of HPLC columns for quantitative studies. To overcome this problem as well as other problems encountered in the prior art, the present invention contemplates a hybrid system, combining microcomputer control of sample injection and data collection, an HPLC pump, and a commercially available cross-linked agarose resin packed in columns as small as 1 mL volume. The present invention is capable of demonstrating interactions of proteins and nucleic acids, antisera and antigen, monoclonal anti-idiotypic antibody and idiotype, as well as autoimmune rheumatoid factor and IgG. The principles of the present invention may be used commercially as a research tool, for quality control of tests involving monoclonal antibodies, and for various other purposes.